Hyaluronic acid composition

ABSTRACT

An injectable hyaluronic acid composition including a hyaluronic acid; a local anesthetic selected from the group of amide and ester type local anesthetics or a combination thereof; and an ascorbic acid derivative in an amount which prevents or reduces the effect on the viscosity and/or elastic modulus G′ of the composition caused by the local anesthetic upon sterilization by heat. Further, the medical and non-medical, such as cosmetic, use of such a composition, and to a method of manufacturing such a composition.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/126,268, filed on Sep. 10, 2018, which is a division of U.S.application Ser. No. 13/983,448, filed on Oct. 2, 2013, now U.S. Pat.No. 10,098,961, which is a U.S. national stage of InternationalApplication No. PCT/EP2012/051875, filed on Feb. 3, 2012, which claimsthe benefit of European Application No. 11153232.1, Feb. 3, 2011. Theentire contents of each of U.S. application Ser. No. 16/126,268, U.S.application Ser. No. 13/983,448, International Application No.PCT/EP2012/051875, and European Application No. 11153232.1 are herebyincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to the field of injectable hyaluronic acidcompositions and the use of such compositions in cosmetic and or medicalapplications.

BACKGROUND

One of the most widely used biocompatible polymers for medical use ishyaluronic acid. It is a naturally occurring polysaccharide belonging tothe group of glycosaminoglycans (GAGs). Hyaluronic acid and the otherGAGs are negatively charged heteropolysaccharide chains which have acapacity to absorb large amounts of water. Hyaluronic acid and productsderived from hyaluronic acid are widely used in the biomedical andcosmetic fields, for instance during viscosurgery and as a dermalfiller.

Water-absorbing gels, or hydrogels, are widely used in the biomedicalfield. They are generally prepared by chemical crosslinking of polymersto infinite networks. While native hyaluronic acid and certaincrosslinked hyaluronic acid products absorb water until they arecompletely dissolved, crosslinked hyaluronic acid gels typically absorba certain amount of water until they are saturated, i.e. they have afinite liquid retention capacity, or swelling degree.

Since hyaluronic acid is present with identical chemical structureexcept for its molecular mass in most living organisms, it gives aminimum of reactions and allows for advanced medical uses. Crosslinkingand/or other modifications of the hyaluronic acid molecule is necessaryto improve its duration in vivo. Furthermore, such modifications affectthe liquid retention capacity of the hyaluronic acid molecule. As aconsequence thereof, hyaluronic acid has been the subject of manymodification attempts.

Hyaluronic acid products for injection are often combined with asuitable anaesthetic, e.g. lidocaine, to reduce pain or discomfortexperienced by the patient due to the injection procedure.

DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide an improvedinjectable hyaluronic acid composition for medical and/or non-medicalapplications.

Hyaluronic acid compositions for use in injection need to be sterilizedbefore use. Sterilization is generally performed by heat treatment, suchas autoclaving. The heat treatment generally leads to a reduction of therigidity or viscosity of the hyaluronic acid composition. As mentionedabove, hyaluronic acid products for injection are often combined with asuitable anaesthetic, e.g. lidocaine, to reduce pain or discomfortexperienced by the patient due to the injection procedure. It has beenobserved that the addition of some commonly used anaesthetics, e.g.lidocaine, counteract the effect of the heat treatment on the rheologyof a hyaluronic acid composition such that the resulting compositionbecomes more rigid or viscous than a gel without lidocaine. This changein rheology may be disadvantageous in some applications, for example inapplications where shallow injection of the gel is required or desired,and/or where a very fine gauge needle is required or desired. Examplesof such applications include skin revitalization and soft tissueaugmentation, for example filling of wrinkles or contouring of the faceor body, by hyaluronic acid gel injections.

It has now been found that addition of a relatively small amount of anascorbic acid derivative to a hyaluronic acid composition comprising alocal anesthetic selected from the group consisting of amide and estertype local anesthetics may effectively reduce the “viscosity increase”of the hyaluronic acid composition caused by the local anesthetic uponsterilization of the composition by autoclave. Thus, the addition of arelatively small amount of an ascorbic acid derivative to a hyaluronicacid composition comprising a local anesthetic may facilitate the use offiner needles for injection without increasing the force required toexpel the composition and without making changes to the hyaluronic acidcomponent. Also, the reduction of the viscosity and/or elastic modulusG′ of the solution is advantageous in applications where the compositionis injected close to the surface of the skin, for example in skinrevitalization or soft tissue augmentation, for example filling ofwrinkles or contouring of the face or body, by hyaluronic acid gelinjection.

The effect of the ascorbic acid derivative on the viscosity and/orelastic modulus G′ on the composition has been shown for both unmodifiedhyaluronic acids and modified, for example crosslinked, hyaluronicacids, which indicates that it is common to all compositions comprisinghyaluronic acid.

Besides the advantageous effect on the viscosity and/or elastic modulusG′ on the composition, the addition of an ascorbic acid derivative tothe composition may also provide further benefits. Ascorbic acid (alsoknown as vitamin C) and its derivatives can act as reducing agents andscavenge aggressive oxidizing agents and radicals. As ascorbic acid andits derivatives can improve the collagen formation, they may enhanceskin morphology. They may also improve epidermal barrier formation,reduce transepidermal water loss, improve wound healing, and thus playan important role in prevention of skin aging and associated dry skinconditions. Ascorbic acid and its derivatives are known for theiranti-inflammatory and photoprotective properties as well as their actionon the improvement of UV-induced skin damage. It has also been shownthat ascorbic acid and its derivatives can clinically improvedermatologic conditions that have inflammation as a component of thedisease process, such as psoriasis and asteototic eczema. As ascorbicacid and its derivatives can suppress the formation of melanin, they mayalso have whitening effect of the skin, and they have been demonstratedto clinically improve melasma and senile freckles. They may also promotehair growth. Ascorbic acid and its derivatives have also been suggestedto have anti-cancer properties.

Addition of an ascorbic acid derivative to the hyaluronic acidcomposition generally has no effect, or little effect, on the stabilityof the composition. Notably, it has been observed the addition of anascorbic acid derivative does not increase the stability of thehyaluronic acid composition. Studies by the inventors have shown thatthe addition of the ascorbic acid derivative may sometimes result in aslight decrease in stability of the hyaluronic acid composition.However, the inventors have found that the advantages associated withadding the ascorbic acid derivative outweigh the slight decrease instability caused by the addition in some cases. In order to avoidunnecessary decrease in stability of the hyaluronic acid composition theconcentration of the ascorbic acid derivative should be kept below themaximum concentrations as set out below.

According to aspects illustrated herein, there is provided an injectablehyaluronic acid composition comprising:

-   -   a hyaluronic acid,    -   a local anesthetic selected from the group consisting of amide        and ester type local anesthetics or a combination thereof, and    -   an ascorbic acid derivative in an amount which prevents or        reduces the effect on the viscosity and/or elastic modulus G′ of        the composition caused by the local anesthetic upon        sterilization by heat.

The term “injectable” means that the hyaluronic acid composition isprovided in a form which is suitable for parenteral injection, e.g. intosoft tissue, such as skin, of a subject or patient. An injectablecomposition should be sterile and free from components that may causeadverse reactions when introduced into soft tissue, such as the skin, ofa subject or patient. This implies that no, or only very mild, immuneresponse occurs in the treated individual. That is, no or only very mildundesirable local or systemic effects occur in the treated individual.

The viscosity and/or elastic modulus G′ of the hyaluronic acidcomposition may be measured according to various methods, well known tothe person skilled in the art. Viscosity may for example be measured asthe “Zero shear viscosity, η₀” by rotational viscometry using a BohlinVOR rheometer (Measuring system C14 or PP 30, Gap 1.00 mm). Othermethods of measuring viscosity may also be applicable. The elasticmodulus G′ may for example be measured using a Bohlin VOR Reometer(Measure system PP 30, Gap 1.00 mm) by performing a strain sweep to findthe linear viscoelastic region (LVR) and then measuring the viscoelasticproperties within the LVR. Other methods of measuring elastic modulus G′may also be applicable.

The injectable hyaluronic acid composition is preferably aqueous and thehyaluronic acid, the local anesthetic and the ascorbic acid derivativeare preferably swelled, dissolved or dispersed in the aqueous phase.

The injectable hyaluronic acid composition comprises a hyaluronic acid.The hyaluronic acid may be a modified, e.g. branched or crosslinked,hyaluronic acid. According to certain embodiments the hyaluronic acid isa crosslinked hyaluronic acid. According to specific embodiments thehyaluronic acid is a hyaluronic acid gel.

Unless otherwise provided, the term “hyaluronic acid” encompasses allvariants and combinations of variants of hyaluronic acid, hyaluronate orhyaluronan, of various chain lengths and charge states, as well as withvarious chemical modifications, including crosslinking. That is, theterm also encompasses the various hyaluronate salts of hyaluronic acidwith various counter ions, such as sodium hyaluronate. Variousmodifications of the hyaluronic acid are also encompassed by the term,such as oxidation, e.g. oxidation of —CH₂OH groups to —CHO and/or —COOH;periodate oxidation of vicinal hydroxyl groups, optionally followed byreduction, e.g. reduction of —CHO to —CH₂OH or coupling with amines toform imines followed by reduction to secondary amines; sulphation;deamidation, optionally followed by deamination or amide formation withnew acids; esterification; crosslinking; substitutions with variouscompounds, e.g. using a crosslinking agent or a carbodiimide assistedcoupling; including coupling of different molecules, such as proteins,peptides and active drug components, to hyaluronic acid; anddeacetylation. Other examples of modifications are isourea, hydrazide,bromocyan, monoepoxide and monosulfone couplings.

The hyaluronic acid can be obtained from various sources of animal andnon-animal origin. Sources of non-animal origin include yeast andpreferably bacteria. The molecular weight of a single hyaluronic acidmolecule is typically in the range of 0.1-10 MDa, but other molecularweights are possible.

In certain embodiments the concentration of said hyaluronic acid is inthe range of 1 to 100 mg/ml. In some embodiments the concentration ofsaid hyaluronic acid is in the range of 2 to 50 mg/ml. In specificembodiments the concentration of said hyaluronic acid is in the range of5 to 30 mg/ml or in the range of 10 to 30 mg/ml. In certain embodiments,the hyaluronic acid is crosslinked. Crosslinked hyaluronic acidcomprises crosslinks between the hyaluronic acid chains, which creates acontinuous network of hyaluronic acid molecules which is held togetherby the covalent crosslinks, physical entangling of the hyaluronic acidchains and various interactions, such as electrostatic interactions,hydrogen bonding and van der Waals forces.

Crosslinking of the hyaluronic acid may be achieved by modification witha chemical crosslinking agent. The chemical crosslinking agent may forexample selected from the group consisting of divinyl sulfone,multiepoxides and diepoxides. According to embodiments the chemicalcrosslinking agent is selected from the group consisting of1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether(EDDE) and diepoxyoctane. According to a preferred embodiment, thechemical crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

The crosslinked hyaluronic acid product is preferably biocompatible.This implies that no, or only very mild, immune response occurs in thetreated individual. That is, no or only very mild undesirable local orsystemic effects occur in the treated individual.

The crosslinked hyaluronic acid product according to the invention maybe a gel, or a hydrogel. That is, it can be regarded as awater-insoluble, but substantially dilute crosslinked system ofhyaluronic acid molecules when subjected to a liquid, typically anaqueous liquid.

The gel contains mostly liquid by weight and can e.g. contain 90-99.9%water, but it behaves like a solid due to a three-dimensionalcrosslinked hyaluronic acid network within the liquid. Due to itssignificant liquid content, the gel is structurally flexible and similarto natural tissue, which makes it very useful as a scaffold in tissueengineering and for tissue augmentation.

As mentioned, crosslinking of hyaluronic acid to form the crosslinkedhyaluronic acid gel may for example be achieved by modification with achemical crosslinking agent, for example BDDE (1,4-butandioldiglycidylether). The hyaluronic acid concentration and the extent ofcrosslinking affects the mechanical properties, e.g. the elastic modulusG′, and stability properties of the gel. Crosslinked hyaluronic acidgels are often characterized in terms of “degree of modification”. Thedegree of modification of hyaluronic acid gels generally range between0.1 and 15 mole %. It has been found that the effect of the ascorbicacid derivative on the viscosity and/or elastic modulus G′ on thecomposition in accordance with the present invention is particularlypronounced in crosslinked hyaluronic acid gels with a low degree ofmodification. The most pronounced effect is obtained in hyaluronic acidgels with a degree of modification of 2 mole % or less, such as 1.5 mole% or less, such as 1.25 mole % or less, for example in the range of 0.1to 2 mole %, such as in the range of 0.2 to 1.5 mole %, such as in therange of 0.3 to 1.25 mole %, as compared to more crosslinked hyaluronicacid gels. The degree of modification (mole %) describes the amount ofcrosslinking agent(s) that is bound to HA, i.e. molar amount of boundcrosslinking agent(s) relative to the total molar amount of repeating HAdisaccharide units. The degree of modification reflects to what degreethe HA has been chemically modified by the crosslinking agent. Reactionconditions for crosslinking and suitable analytical techniques fordetermining the degree of modification are all well known to the personskilled in the art, who easily can adjust these and other relevantfactors and thereby provide suitable conditions to obtain a degree ofmodification in the range of 0.1-2% and verify the resulting productcharacteristics with respect to the degree of modification. A BDDE(1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel may forexample be prepared according to the method described in Examples 1 and2 of published international patent application WO 9704012.

In a preferred embodiment the hyaluronic acid of the composition ispresent in the form of a crosslinked hyaluronic acid gel crosslinked bya chemical crosslinking agent, wherein the concentration of saidhyaluronic acid is in the range of 10 to 30 mg/ml and the degree ofmodification with said chemical crosslinking agent is in the range of0.1 to 2 mole %.

Hyaluronic acid gels may also comprise a portion of hyaluronic acidwhich is not crosslinked, i.e not bound to the three-dimensionalcrosslinked hyaluronic acid network. However, it is preferred that atleast 50% by weight, preferably at least 60% by weight, more preferablyat least 70% by weight, and most preferably at least 80% by weight, ofthe hyaluronic acid in a gel composition form part of the crosslinkedhyaluronic acid network.

The injectable hyaluronic acid composition further comprises a localanesthetic selected from the group consisting of amide and ester typelocal anesthetics or a combination thereof. A local anesthetic is a drugthat causes reversible local anesthesia and a loss of nociception. Whenit is used on specific nerve pathways (nerve block), effects such asanalgesia (loss of pain sensation) and paralysis (loss of muscle power)can be achieved. The local anesthetic may be added to the hyaluronicacid composition to reduce pain or discomfort experienced by the patientdue to the injection procedure. The groups of amide (also commonlyreferred to as aminoamide) type local anesthetics and ester (alsocommonly referred to as aminoester) type local anesthetics are welldefined and recognized in the art.

Amide and ester type local anesthetic molecules are built on a simplechemical plan, consisting of an aromatic part linked by an amide orester bond to a basic side-chain. The only exception is benzocaine whichhas no basic group. All other anesthetics are weak bases, with pKavalues mainly in the range 8-9, so that they are mainly but notcompletely, ionized at physiological pH. As a result of their similaritythey may be expected to have similar chemical and physical effects onthe hyaluronic acid composition.

According to certain embodiments the local anesthetic is selected fromthe group consisting of amide and ester type local anesthetics, forexample bupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine),clibucaine, ethyl parapiperidinoacetylaminobenzoate, etidocaine,lignocaine (lidocaine), mepivacaine, oxethazaine, prilocaine,ropivacaine, tolycaine, trimecaine, vadocaine, articaine,levobupivacaine, amylocaine, cocaine, propanocaine, clormecaine,cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine,butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine,dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine,procaine (novocaine), propoxycaine, tricaine or a combination thereof.

According to certain embodiments the local anesthetic is selected fromthe group consisting amide type local anesthetics, for examplebupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine),clibucaine, ethyl parapiperidinoacetylaminobenzoate, etidocaine,lignocaine (lidocaine), mepivacaine, oxethazaine, prilocaine,ropivacaine, tolycaine, trimecaine, vadocaine, articaine,levobupivacaine or a combination thereof. According to some embodimentsthe local anesthetic is selected from the group consisting ofbupivacaine, lidocaine, and ropivacaine, or a combination thereof.According to specific embodiments the local anesthetic is lidocaine.Lidocaine is a well-known substance, which has been used extensively asa local anesthetic in injectable formulations, such as hyaluronic acidcompositions.

The concentration of the amide or ester local anesthetic may be selectedby the skilled person within the therapeutically relevant concentrationranges of each specific local anesthetic or a combination thereof.

In certain embodiments the concentration of said local anesthetic is inthe range of 0.1 to 30 mg/ml. In some embodiments the concentration ofsaid local anesthetic is in the range of 0.5 to 10 mg/ml.

When lidocaine is used as the local anesthetic, the lidocaine maypreferably be present in a concentration in the range of 1 to 5 mg/ml,more preferably in the range of 2 to 4 mg/ml, such as in a concentrationof about 3 mg/ml.

The injectable hyaluronic acid composition further comprises an ascorbicacid derivative. The term “ascorbic acid derivative”, as used herein,means ascorbic acid or derivatives of ascorbic acid comprising thegeneral chemical structure of ascorbic acid. Thus, the ascorbic acidderivative may be a compound comprising the chemical structure:

The ascorbic acid derivative of the composition may be ascorbic acid ora compound structurally related to and/or derived from ascorbic acid.Ascorbic acid itself may be useful in some applications, but because ofits low stability it may be of limited use in some practicalapplications.

The ascorbic acid derivative may be water soluble. The solubility of theascorbic acid derivative in water under atmospheric conditions maypreferably be sufficient to allow dissolution of a desired concentrationof the ascorbic acid derivative in the composition. The solubility ofthe water soluble ascorbic acid derivative in water under atmosphericconditions may preferably be sufficient to allow a concentration of0.001 mg/ml or more, and more preferably 0.01 mg/ml or more, in thehyaluronic acid composition.

The ascorbic acid derivative may be capable of forming ascorbic acid orascorbate in vivo, for example through enzymatic degradation mediated byphosphatases, glucosidases, etc. Thus, according to an embodiment, theascorbic acid derivative is capable of forming ascorbic acid orascorbate when placed in in vivo conditions.

In some embodiments, the ascorbic acid derivative is selected from thegroup consisting of a phosphate ester of ascorbic acid, a carboxylicacid ester of ascorbic acid, a sulfate of ascorbic acid, a sulfonateester of ascorbic acid, a carbonate of ascorbic acid and an acetal orketal substituted ascorbic acid, or a combination thereof.

The ascorbic acid derivative may, for example, be a compound having thegeneral formula:

wherein R1, R2, R3, R4 are, independent of each other, H or an organicsubstituent. Compound I may for example be a phosphate ester of ascorbicacid, whereinat least one of R1, R2, R3 and R4 is

where X is H, alkyl, alkenyl, alkynyl, aryl, an amine, an alcohol, aglycoside, or

where n can be 0 to 500.

Counter ions can be, but are not limited to, Na⁺, K⁺, Ca²⁺, Al³⁺, Li⁺,Zn²⁺ or Mg²⁺.

Compound I may for example be a carboxylic acid ester of ascorbic acid,wherein at least one of R1, R2, R3 and R4 is

where Y is H, alkyl, alkenyl, alkynyl, aryl, an amine, an alcohol, aglycoside, an amino acid ester or

where n can be 0 to 500.

Compound I may for example be a sulfate of ascorbic acid, wherein atleast one of R1, R2, R3 and R4 is

Counter ions can be, but are not limited to, Na⁺, K⁺, Ca²⁺, Al³⁺, Li⁺,Zn²⁺ or Mg²⁺.

Compound I may for example be a sulfonate ester of ascorbic acid,wherein at least one of R1, R2, R3 and R4 is

where Z is H, alkyl, alkenyl, alkynyl, aryl, an amine, an alcohol, aglycoside, or

where n can be 0 to 500.

Compound I may for example be a carbonate of ascorbic acid, wherein atleast one of R1, R2, R3 and R4 is

where U is H, alkyl, alkenyl, alkynyl, aryl, an amine, an alcohol, aglycoside, or

where n can be 0 to 500.

Compound I may for example be an acetal or ketal substituted ascorbicacid, wherein at least one of R1, R2, R3 and R4 is

where W, W′, and W″ are H, alkyl, alkenyl, alkynyl, aryl, an amine, analcohol, or a carbohydrate residue, for example:

Compound I may for example be an acetal or ketal substituted ascorbicacid having the general formula:

where H is hydrogen, Alk is alkyl and Ar is aryl.

In some embodiments, the ascorbic acid derivative is selected from thegroup consisting of ascorbyl phosphates, ascorbyl sulfates, and ascorbylglycosides, or a combination thereof.

In certain embodiments the ascorbic acid derivative is selected from thegroup consisting of ascorbyl phosphates and ascorbyl glycosides, or acombination thereof.

In some embodiments the ascorbyl phosphate is selected from the groupconsisting of sodium ascorbyl phosphate (SAP) and magnesium ascorbylphosphate (MAP), or a combination thereof. Ascorbyl phosphates convertto vitamin C in vivo by enzymatic hydrolysis by phosphatases.

In some embodiments, the ascorbic acid derivative is an aminoalkylascorbyl phosphate. In certain embodiments, the ascorbic acid derivativeis aminopropyl ascorbyl phosphate.

In some embodiments, the ascorbic acid derivative is ascorbyl glucoside.Ascorbyl glucoside converts to vitamin C in vivo by enzymatic hydrolysisby glucosidases.

In some embodiments, the ascorbic acid derivative is methylsilanolascorbate.

In some embodiments, the ascorbic acid derivative is L-ascorbic acidacetonide.

The ascorbic acid derivatives described herein may be in unprotonated orfully or partially protonated form, or in the form a pharmaceuticallyacceptable salt. Specifically, the terms ascorbyl phosphate, ascorbylsulfate, aminoalkyl ascorbyl phosphate, aminopropyl ascorbyl phosphate,ascorbyl glycoside and ascorbyl glucoside, as used herein, are intendedto encompass the compounds in unprotonated or fully or partiallyprotonated form, or in the form a pharmaceutically acceptable salt.Examples of suitable counter ions include, but are not limited to,aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

The concentration of the ascorbic acid derivative may be selected by theskilled person depending on the specific ascorbic acid derivative used.

In certain embodiments the concentration of said ascorbic acidderivative is in the range of 0.001 to 15 mg/ml. In certain embodimentsthe concentration of said ascorbic acid derivative is in the range of0.001 to 10 mg/ml. In some embodiments the concentration of saidascorbic acid derivative is in the range of 0.01 to 5 mg/ml. Aconcentration of said ascorbic acid derivative of above 0.01 mg/ml ispreferred since it provides a more marked reduction in viscosity and/orelastic modulus G′ of the hyaluronic acid composition. A concentrationof said ascorbic acid derivative of below 5 mg/ml is preferred sincehigher concentrations may result in unnecessary decrease of stability ofthe hyaluronic acid composition without additional benefits.

The required concentration of the ascorbic acid derivative may varywithin the above specified ranges depending on the particular ascorbicacid derivative used. As an example, a suitable concentration of sodiumascorbyl phosphate (SAP) or magnesium ascorbyl phosphate (MAP) may be inthe range of 0.01 to 1 mg/ml, while a suitable concentration of ascorbylglucoside may be in the range of 0.1 to 5 mg/ml.

Thus, according to an embodiment, the ascorbic acid derivative is sodiumascorbyl phosphate (SAP) or magnesium ascorbyl phosphate (MAP) in aconcentration in the range of 0.01 to 1 mg/ml and preferably in therange of 0.01 to 0.5 mg/ml.

According to another embodiment, the ascorbic acid derivative isascorbyl glucoside in a concentration in the range of 0.01 to 1 mg/ml,preferably in the range of 0.01 to 0.8 mg/ml, and more preferably in therange of 0.05 to 0.4 mg/ml.

As mentioned, it has been observed the addition of an ascorbic acidderivative does not increase the stability of the hyaluronic acidcomposition. In other words, the injectable hyaluronic acid compositionaccording to the present invention does not exhibit increased stabilitycompared to the same composition without an ascorbic acid derivative.

The term stability, as used herein, is used to denote the ability of thehyaluronic acid composition to resist degradation during storage andhandling prior to use. It is known that the addition of constituents toa hyaluronic acid or hyaluronic acid gel may affect the stability ofsaid hyaluronic acid or hyaluronic acid gel. Stability of hyaluronicacid or hyaluronic acid gel composition can be determined by a range ofdifferent methods. Methods for determining stability include, but arenot limited to, assessing homogeneity, color, clarity, pH, gel contentand rheological properties of the composition. Stability of a hyaluronicacid composition is often determined by observing or measuring one ormore of said parameters over time. Stability may for example bedetermined by measuring the viscosity and/or elastic modulus G′ of thehyaluronic acid composition over time. Viscosity may for example bemeasured as the “Zero shear viscosity, η₀” by rotational viscometryusing a Bohlin VOR rheometer (Measuring system C14 or PP 30, Gap 1.00mm). Other methods of measuring viscosity may also be applicable. Theelastic modulus G′ may for example be measured using a Bohlin VORReometer (Measure system PP 30, Gap 1.00 mm) by performing a strainsweep to find the linear viscoelastic region (LVR) and then measuringthe viscoelastic properties within the LVR. Other methods of measuringelastic modulus G′ may also be applicable.

In a more specific embodiment, there is provided an injectablehyaluronic acid composition, comprising: an aqueous hyaluronic acid gelcomprising 2 to 50 mg/ml of a hyaluronic acid; 0.5 to 10 mg/ml oflidocaine; and 0.01 to 5 mg/ml of an ascorbic acid derivative selectedfrom the group consisting of ascorbyl phosphates and ascorbylglycosides, or a combination thereof.

In a more specific embodiment, there is provided an injectablehyaluronic acid composition, comprising: an aqueous hyaluronic acid gelcomprising 2 to 50 mg/ml of a hyaluronic acid; 0.5 to 10 mg/ml oflidocaine; and 0.01 to 5 mg/ml of an ascorbyl phosphate, for examplesodium or magnesium ascorbyl phosphate.

In another more specific embodiment, there is provided an injectablehyaluronic acid composition, comprising: an aqueous hyaluronic acid gelcomprising 2 to 50 mg/ml of a hyaluronic acid; 0.5 to 10 mg/ml oflidocaine; and 0.01 to 5 mg/ml of an ascorbyl glycoside, for exampleascorbyl glucoside.

In some embodiments, the composition has been subjected tosterilization. In certain embodiments is the composition sterilized,i.e. the composition has been subjected to heat and/or steam treatmentin order to sterilize the composition. In some embodiments thecomposition has been subjected to sterilization by autoclaving orsimilar sterilization by heat or steam. Sterilization, e.g. autoclaving,may be performed at a F₀-value≥4. The F₀ value of a saturated steamsterilisation process is the lethality expressed in terms of theequivalent time in minutes at a temperature of 121° C. delivered by theprocess to the product in its final container with reference tomicro-organisms posessing a Z-value of 10.

When hyaluronic acid compositions are subjected to sterilization bytreatment with heat or steam, the viscosity and/or elastic modulus G′are generally reduced. When an amide or ester type local anesthetic isadded to the hyaluronic acid composition, this reduction in viscosityand/or elastic modulus G′ is decreased, resulting in a firmer or moreviscous final product. The addition of the ascorbic acid derivativecounteracts this effect of the local anesthetic, thereby producing afinal product, having a viscosity and/or elastic modulus G′ more closelyresembling those of the hyaluronic acid composition without the localanesthetic, without making changes to the hyaluronic acid component.

The crosslinked hyaluronic acid product according to the invention, oran aqueous composition thereof, may be provided in the form of apre-filled syringe, i.e. a syringe that is pre-filled with a crosslinkedhyaluronic acid composition and autoclaved.

The injectable hyaluronic acid compositions described herein may beemployed in medical as well as non-medical, e.g. purely cosmetic,procedures by injection of the composition into soft tissues of apatient or subject. The compositions have been found useful in, e.g.,soft tissue augmentation, for example filling of wrinkles, by hyaluronicacid gel injection. The compositions have been found especially usefulin a cosmetic treatment, referred to herein as skin revitalization,whereby small quantities of the hyaluronic acid composition are injectedinto the dermis at a number of injection sites distributed over an areaof the skin to be treated, resulting in improved skin tone and skinelasticity. Skin revitalization is a simple procedure and health risksassociated with the procedure are very low. According to other aspectsillustrated herein, there is provided an injectable hyaluronic acidcomposition as described above for use as a medicament. The compositionis useful, for example in the treatment of various dermatologicalconditions. Particularly, there is provided an injectable hyaluronicacid composition as described above for use in a dermatologicaltreatment selected from the group consisting of wound healing, treatmentof dry skin conditions or sun-damaged skin, treatment of hyperpigmentation disorders, treatment and prevention of hair loss, andtreatment of conditions that have inflammation as a component of thedisease process, such as psoriasis and asteototic eczema. In otherwords, there is provided an injectable hyaluronic acid composition asdescribed above for use in the manufacture of a medicament for use in adermatological treatment selected from the group consisting of woundhealing, treatment of dry skin conditions or sun-damaged skin, treatmentof hyper pigmentation disorders, treatment and prevention of hair loss,and treatment of conditions that have inflammation as a component of thedisease process, such as psoriasis and asteototic eczema.

According to another embodiment there is provided an injectablehyaluronic acid composition as described above for use in the treatmentof a joint disorder by intraarticular injection.

According to other aspects illustrated herein, there is provided the useof an injectable hyaluronic acid composition as described above forcosmetic, non-medical, treatment of a subject by injection of thecomposition into the skin of the subject. A purpose of the cosmetic,non-medical, treatment may be for improving the appearance of the skin,preventing and/or treating hair loss, filling wrinkles or contouring theface or body of a subject. The cosmetic, non-medical, use does notinvolve treatment of any form of disease or medical condition. Examplesof improving the appearance of the skin include, but are not limited to,treatment of sun-damaged or aged skin, skin revitalization, skinwhitening and treatment of hyper pigmentation disorders such as senilefreckles, melasma and ephelides.

According to certain embodiments, there is provided the use of aninjectable hyaluronic acid composition as described above for improvingthe appearance of skin, preventing and/or treating hair loss, fillingwrinkles or contouring the face or body of a subject. The use preferablycomprises injecting the composition into the cutis and/or subcutis of ahuman subject. The use of the injectable hyaluronic acid composition forimproving the appearance of skin, preventing and/or treating hair loss,filling wrinkles or contouring the face or body of a subject, may beessentially or totally non-medical, e.g. purely cosmetic.

According to certain embodiments, there is provided the use of aninjectable hyaluronic acid composition as described above for improvingthe appearance of skin. According to a preferred embodiment, there isprovided the use of an injectable hyaluronic acid composition asdescribed above for skin revitalization.

According to certain embodiments, there is provided the use of aninjectable hyaluronic acid composition as described above for preventingand/or treating hair loss.

According to certain embodiments, there is provided the use of aninjectable hyaluronic acid composition as described above for fillingwrinkles or contouring the face or body of a subject.

According to other aspects illustrated herein, there is provided amethod of improving the appearance of skin, preventing and/or treatinghair loss, filling wrinkles or contouring the face or body of a subject,comprising:

a) providing an injectable hyaluronic acid composition as describedabove, and

b) injecting said injectable hyaluronic acid composition into the skinof a subject.

In certain embodiments the injectable hyaluronic acid composition isinjected into the cutis and/or subcutis.

According to certain embodiments, the method comprises improving theappearance of skin. According to a preferred embodiment, the methodcomprises skin revitalization.

According to certain embodiments, the method comprises preventing and/ortreating hair loss.

According to certain embodiments, the method comprises filling wrinklesor contouring the face or body of a subject.

According to other aspects illustrated herein, there is provided amethod of manufacturing a hyaluronic acid composition comprising:

a) mixing a hyaluronic acid, a local anesthetic selected from the groupconsisting of amide and ester type local anesthetics or a combinationthereof, and an ascorbic acid derivative in an amount which prevents orreduces the effect on the viscosity and/or elastic modulus G′ of thecomposition caused by the local anesthetic upon sterilization by heat,and

b) subjecting the mixture to sterilization by heat.

In the method of manufacturing the composition, said ascorbic acidderivative is operative for preventing or reducing the effect of thelocal anesthetic on the viscosity and/or elastic modulus G′ of thecomposition due to the sterilization by heat.

The components of the composition, i.e. hyaluronic acid, localanesthetic and ascorbic acid derivative, may be further defined asdescribed above for the injectable hyaluronic acid composition.

The manufactured hyaluronic acid composition does not exhibit increasedstability compared to the same composition without an ascorbic acidderivative.

In some embodiments, the sterilization of step b) comprises subjectingthe mixture to a heat treatment. In certain embodiments, thesterilization of step b) comprises autoclaving the mixture at aF₀-value≥4. The sterilization may be further characterized as describedabove for the composition.

According to other aspects illustrated herein, there is provided the useof an ascorbic acid derivative in an injectable hyaluronic acidcomposition, further comprising a hyaluronic acid and a local anestheticselected from the group consisting of amide and ester type localanesthetics or a combination thereof, for preventing or reducing theeffect of the local anesthetic on the viscosity and/or elastic modulusG′ of the composition due to the sterilization by heat.

The components of the composition may be further defined as describedabove for the injectable hyaluronic acid composition. The sterilizationmay be further characterized as described above.

The injectable hyaluronic acid composition formed by use of an ascorbicacid derivative does not exhibit increased stability compared to thesame composition without an ascorbic acid derivative.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of MAP (Magnesium AscorbylPhosphate) on a hyaluronic acid gel with lidocaine.

FIG. 2 is a graph showing the effect of MAP on a hyaluronic acid gelwith lidocaine.

FIG. 3 is a graph showing the effect of MAP on a non-crosslinkedhyaluronic acid with lidocaine.

FIG. 4 is a graph showing the effect of MAP on a non-crosslinkedhyaluronic acid with lidocaine.

FIG. 5 is a graph showing the effect of MAP on a hyaluronic acid gelwith lidocaine autoclaved at various F₀ values.

FIG. 6 is a graph showing the effect of MAP on a hyaluronic acid gelwith bupivacaine.

FIG. 7 is a graph showing the effect of MAP on a hyaluronic acid gelwith tetracaine.

FIG. 8 is a graph showing the effect of SAP (Sodium Ascorbyl Phosphate)on a hyaluronic acid gel with lidocaine.

FIG. 9 is a graph showing the effect of Methylsilanol ascorbate on ahyaluronic acid gel with lidocaine.

FIG. 10 is a graph showing the effect of Ascorbyl glucoside on anon-crosslinked hyaluronic acid with bupivacaine.

FIG. 11 is a graph showing the effect of different concentrations of SAPon a hyaluronic acid gel with lidocaine.

FIG. 12 is a graph showing the effect of L-Ascorbic acid acetonide on ahyaluronic acid gel with tetracaine.

FIG. 13 is a graph showing the effect of SAP on a hyaluronic acid gelwith lidocaine.

FIG. 14 is a graph showing the effect of Aminopropyl Ascorbyl Phosphateon a non-crosslinked hyaluronic acid with lidocaine.

FIG. 15 is a graph showing the effect of Ascorbyl glucoside on ahyaluronic acid gel with lidocaine.

FIG. 16 is a graph showing the effect of Ascorbyl glucoside on ahyaluronic acid gel with lidocaine.

FIG. 17 is a graph showing the effect of Ascorbyl glucoside on ahyaluronic acid gel with lidocaine.

FIG. 18 is a graph showing the effect of MAP on a hyaluronic acid gelswith lidocaine.

FIG. 19 is a graph showing the effect of SAP on a hyaluronic acid gelwith lidocaine in a stability study.

FIG. 20 is a graph showing the effect of Ascorbyl glucoside on ahyaluronic acid gel with lidocaine in a stability study.

FIG. 21 is a graph showing the effect of MAP or Ascorbyl glucoside on ahyaluronic acid gel with lidocaine in a stability study.

FIG. 22 is a graph showing the effect of Ascorbyl glucoside on ahyaluronic acid gel with lidocaine in a stability study.

ITEMIZED LISTING OF EMBODIMENTS

1. An injectable hyaluronic acid composition comprising

-   -   a hyaluronic acid,    -   a local anesthetic selected from the group consisting of amide        and ester type local anesthetics or a combination thereof, and    -   an ascorbic acid derivative in an amount which prevents or        reduces the effect on the viscosity and/or elastic modulus G′ of        the composition caused by the local anesthetic upon        sterilization by heat.

2. An injectable hyaluronic acid composition according to item 1,wherein said composition does not exhibit increased stability comparedto the same composition without an ascorbic acid derivative.

3. An injectable hyaluronic acid composition according to any one of thepreceding items, wherein said hyaluronic acid is a modified hyaluronicacid.

4. An injectable hyaluronic acid composition according to item 3,wherein said hyaluronic acid is a hyaluronic acid gel.

5. An injectable hyaluronic acid composition according to item 4,wherein the hyaluronic acid gel is crosslinked by modification with achemical crosslinking agent.

6. An injectable hyaluronic acid composition according to item 5,wherein the chemical crosslinking agent is selected from the groupconsisting of divinyl sulfone, multiepoxides and diepoxides.

7. An injectable hyaluronic acid composition according to item 6,wherein the chemical crosslinking agent is selected from the groupconsisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanedioldiglycidyl ether (EDDE) and diepoxyoctane.

8. An injectable hyaluronic acid composition according to item 7,wherein the chemical crosslinking agent is 1,4-butanediol diglycidylether (BDDE).

9. An injectable hyaluronic acid composition according to any one ofitems 5-8, wherein the hyaluronic acid gel has a degree of modificationof 2 mole % or less, such as 1.5 mole % or less, such as 1.25 mole % orless.

10. An injectable hyaluronic acid composition according to any one ofitems 5-8, wherein the hyaluronic acid gel has a degree of modificationin the range of 0.1 to 2 mole %, such as in the range of 0.2 to 1.5 mole%, such as in the range of 0.3 to 1.25 mole %.

11. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein the concentration of said hyaluronic acidis in the range of 1 to 100 mg/ml.

12. An injectable hyaluronic acid composition according to item 11,wherein the concentration of said hyaluronic acid is in the range of 2to 50 mg/ml.

13. An injectable hyaluronic acid composition according to item 12,wherein the concentration of said hyaluronic acid is in the range of 10to 30 mg/ml.

14. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said local anesthetic is selected from thegroup consisting of lignocaine (lidocaine), bupivacaine, butanilicaine,carticaine, cinchocaine (dibucaine), clibucaine,ethylparapiperidinoacetylaminobenzoate, etidocaine, mepivacaine,oxethazaine, prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine,articaine, levobupivacaine, amylocaine, cocaine, propanocaine,clormecaine, cyclomethycaine, proxymetacaine, amethocaine (tetracaine),benzocaine, butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine,dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine,procaine (novocaine), propoxycaine, tricaine, or a combination thereof.

15. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said local anesthetic is selected from thegroup consisting of amide type local anesthetics, or a combinationthereof.

16. An injectable hyaluronic acid composition according to item 15,wherein said local anesthetic is selected from the group consisting oflignocaine (lidocaine), bupivacaine, butanilicaine, carticaine,cinchocaine (dibucaine), clibucaine, ethylparapiperidinoacetylaminobenzoate, etidocaine, mepivacaine, oxethazaine,prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine, articaine,levobupivacaine or a combination thereof.

17. An injectable hyaluronic acid composition according to item 16,wherein said local anesthetic is selected from the group consisting oflidocaine, bupivacaine, and ropivacaine, or a combination thereof.

18. An injectable hyaluronic acid composition according to item 17,wherein said local anesthetic is lidocaine.

19. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein the concentration of said local anestheticis in the range of 0.1 to 30 mg/ml.

20. An injectable hyaluronic acid composition according to item 19,wherein the concentration of said local anesthetic is in the range of0.5 to 10 mg/ml.

21. An injectable hyaluronic acid composition according to item 20,wherein the concentration of said lidocaine is in the range of 1 to 5mg/ml.

22. An injectable hyaluronic acid composition according to item 21,wherein the concentration of said lidocaine is in the range of 2 to 4mg/ml.

23. An injectable hyaluronic acid composition according to item 22,wherein the concentration of said lidocaine is about 3 mg/ml.

24. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said ascorbic acid derivative is a compoundcomprising the chemical structure:

25. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said ascorbic acid derivative is watersoluble under atmospheric conditions.

26. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said ascorbic acid derivative is capable offorming ascorbic acid or ascorbate when placed in in vivo conditions.

27. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said ascorbic acid derivative is selectedfrom the group consisting of ascorbyl phosphates, ascorbyl sulfates, andascorbyl glycosides, or a combination thereof.

28. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said ascorbic acid derivative is selectedfrom the group consisting of ascorbyl phosphates and ascorbylglycosides, or a combination thereof.

29. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein said ascorbic acid derivative is anascorbyl phosphate.

30. An injectable hyaluronic acid composition according to item 29,wherein said ascorbyl phosphate is selected from the group consisting ofsodium ascorbyl phosphate (SAP) and magnesium ascorbyl phosphate (MAP).

31. An injectable hyaluronic acid composition according to any one ofitems 1-28, wherein said ascorbic acid derivative is an ascorbylglycoside.

32. An injectable hyaluronic acid composition according to item 31,wherein said ascorbic acid derivative is ascorbyl glucoside.

33. An injectable hyaluronic acid composition according to any one ofthe preceding items, wherein the concentration of said ascorbic acidderivative is in the range of 0.001 to 15 mg/ml.

34. An injectable hyaluronic acid composition according to item 33,wherein the concentration of said ascorbic acid derivative is in therange of 0.001 to 10 mg/ml.

35. An injectable hyaluronic acid composition according to item 34,wherein the concentration of said ascorbic acid derivative is in therange of 0.01 to 5 mg/ml.

36. An injectable hyaluronic acid composition according to item 35,wherein the concentration of said ascorbic acid derivative is in therange of 0.01 to 0.5 mg/ml.

37. An injectable hyaluronic acid composition according to item 30,wherein the concentration of said sodium ascorbyl phosphate (SAP) ormagnesium ascorbyl phosphate (MAP) is in the range of 0.01 to 1 mg/ml.

38. An injectable hyaluronic acid composition according to item 37,wherein the concentration of said sodium ascorbyl phosphate (SAP) ormagnesium ascorbyl phosphate (MAP) is in the range of 0.01 to 0.5 mg/ml.

39. An injectable hyaluronic acid composition according to item 31,wherein the concentration of said ascorbyl glucoside is in the range of0.01 to 0.8 mg/ml.

40. An injectable hyaluronic acid composition according to item 39,wherein the concentration of said ascorbyl glucoside is in the range of0.05 to 0.4 mg/ml.

41. An injectable hyaluronic acid composition according to item 1,comprising

-   -   an aqueous hyaluronic acid gel comprising 2 to 50 mg/ml of a        hyaluronic acid,    -   0.5 to 10 mg/ml of lidocaine, and    -   0.01 to 5 mg/ml of an ascorbic acid derivative selected from the        group consisting of ascorbyl phosphates and ascorbyl glycosides,        or a combination thereof.

42. A sterilized injectable hyaluronic acid composition according to anyone of the preceding items.

43. A sterilized hyaluronic acid composition according to item 42,wherein the composition has been subjected to sterilization byautoclaving or similar sterilization by heat.

44. An injectable hyaluronic acid composition as defined in any one ofitems 1-43 for use as a medicament.

45. An injectable hyaluronic acid composition as defined in any one ofitems 1-43 for use in a dermatological treatment selected from the groupconsisting of wound healing, treatment of dry skin conditions andsun-damaged skin, treatment of hyper pigmentation disorders, treatmentand prevention of hair loss, and treatment of conditions that haveinflammation as a component of the disease process, such as psoriasisand asteototic eczema.

46. An injectable hyaluronic acid composition as defined in any one ofitems 1-43 for use in the treatment of a joint disorder byintraarticular injection.

47. Cosmetic, non-medical use of an injectable acid composition asdefined in any one of items 1-43 for improving the appearance of skin,preventing and/or treating hair loss, filling wrinkles or contouring theface or body of a subject.

48. Cosmetic, non-medical use according to item 47, for improving theappearance of the skin of a subject.

49. Cosmetic, non-medical use according to item 47, for filling wrinklesof a subject.

50. Cosmetic, non-medical method of improving the appearance of skin,preventing and/or treating hair loss, filling wrinkles or contouring theface or body of a subject, comprising

-   -   a) providing an injectable hyaluronic acid composition as        defined in any one of items 1-43, and    -   b) injecting said injectable hyaluronic acid composition into        the skin of a subject.

51. A method according to item 50, wherein said injectable hyaluronicacid composition is injected into the cutis and/or subcutis.

52. A method of manufacturing a hyaluronic acid composition comprising:

-   -   a) mixing a hyaluronic acid, a local anesthetic selected from        the group consisting of amide and ester type local anesthetics        or a combination thereof, and an ascorbic acid derivative in an        amount which prevents or reduces the effect on the viscosity        and/or elastic modulus G′ of the composition caused by the local        anesthetic upon sterilization by heat, and    -   b) subjecting the mixture to sterilization by heat.

53. A method according to item 52, wherein the formed hyaluronic acidcomposition does not exhibit increased stability compared to the samecomposition without an ascorbic acid derivative.

54. A method according any one of items 52 and 53, wherein step b)comprises subjecting the mixture to a F₀-value≥4.

55. Use of an ascorbic acid derivative in an injectable hyaluronic acidcomposition further comprising

-   -   a hyaluronic acid and    -   a local anesthetic selected from the group consisting of amide        and ester type local anesthetics or a combination thereof,        for preventing or reducing the effect of the local anesthetic on        the viscosity and/or elastic modulus G′ of the composition due        to sterilization by heat.

56. Use according to item 55, wherein the hyaluronic acid compositiondoes not exhibit increased stability compared to the same compositionwithout an ascorbic acid derivative.

EXAMPLES

Without desiring to be limited thereto, the present invention will inthe following be illustrated by way of examples. Since hyaluronic acidpolymer and hyaluronic acid gel may always be subject to some batch tobatch variations, each example has been performed with a single batch ofhyaluronic acid polymer or hyaluronic acid gel in order to obtaincomparable results. Slight variations in, e.g., rheological propertiesor viscosity between similar compositions in different examples may bedue to such batch to batch variations.

Example 1. Hyaluronic Acid Gel with Lidocaine and MAP

In this experiment, the rheological properties after autoclaving ofhyaluronic acid gels without additives were compared to hyaluronic acidgels with added lidocaine and hyaluronic acid gels with added lidocaineand MAP respectively.

Formulations having various concentrations lidocaine and MAP as outlinedin Table 1 were prepared as described below.

TABLE 1 G′ Formulation HA Gel Lidocaine MAP at 1.0 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 1a 20 0 0 239 1b 20 3 0 437 1c 20 3 0.07 394 1d 20 3 0.7211 1e 20 1 0 440 1f 20 1 0.07 388 1g 20 1 0.7 206

In all formulations a BDDE (1,4-butandiol diglycidylether) crosslinkedhyaluronic acid gel with a degree of modification of 1 mole % and ahyaluronic acid content of 20 mg/ml was used. The degree of modification(mole %) describes the amount of crosslinking agent(s) that is bound toHA, i.e. molar amount of bound crosslinking agent(s) relative to thetotal molar amount of repeating HA disaccharide units. The degree ofmodification reflects to what degree the HA has been chemically modifiedby the crosslinking agent.

The BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gelmay for example be prepared according to the method described inExamples 1 and 2 of published international patent application WO9704012.

A stock-solution of lidocaine hydrochloride monohydrate (CAS number6108-05-0, Sigma Aldrich, St Louis, USA) was prepared by dissolvinglidocaine hydrochloride monohydrate in WFI (water for injection) and astock-solution of Magnesium Ascorbyl Phosphate (MAP, CAS number114040-31-2, Nikko Chemicals co, Japan), was prepared by dissolving MAPin phosphate buffered saline (8 mM, 0.9% NaCl).

Formulation 1a:

The hyaluronic acid gel was diluted to the same degree as 1b-1g byadding phosphate buffered saline (8 mM, 0.9% NaCl).

Formulation 1b:

Stock-solution of lidocaine was added to the hyaluronic acid gel to afinal concentration of 3 mg/ml gel.

Formulation 1c:

Stock-solution of lidocaine and stock-solution of MAP were added to thehyaluronic acid gel to the final concentrations of 3 mg lidocaine/ml and0.07 mg MAP/ml gel.

Formulations 1d-1g were prepared in the same manner by varying theamounts of lidocaine stock-solution and MAP stock-solution. To allformulations phosphate buffered saline (8 mM, 0.9% NaCl) was added toadjust the dilution to the same degree.

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜30).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 1. MAP counteracts the effect on theelastic modulus G′ of the composition caused by the local anestheticupon sterilization by heat. The higher the concentration of MAP thelarger is the decrease on the elastic G′ modulus. A higher concentrationof lidocaine does not affect the increase on the elastic modulus G′.

Example 2. Hyaluronic Acid Gel with a Higher Degree of Modification withLidocaine and MAP

Formulations as outlined in Table 2 were prepared essentially accordingto the method described in Example 1, with the exception that ahyaluronic acid gel with a higher degree of modification (approximately7%) was used. The hyaluronic acid gel may for example be preparedaccording to the method described in the examples of U.S. Pat. No.6,921,819 B2.

TABLE 2 G′ Formulation HA Gel Lidocaine MAP at 1.0 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 2a 20 0 0 393 2b 20 3 0 417 2c 20 3 0.3 388

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜29).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 2. MAP counteracts the effect on theelastic modulus G′ of the composition caused by the local anestheticupon sterilization by heat.

Example 3. Non-Crosslinked Hyaluronic Acid with Lidocaine and MAP

Formulations as outlined in Table 3 were prepared essentially accordingto the method described in Example 1, with the exception that anon-crosslinked hyaluronic acid with an average molecular weight of1×10⁶ Da was used.

TABLE 3 Zero shear Formulation HA Lidocaine MAP viscosity # [mg/ml][mg/ml] [mg/ml] η₀ [Pas] 3a 20 0 0 3.83 3b 20 3 0 4.26 3c 20 3 0.07 2.453d 20 3 0.3 1.98

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The viscosity of the formulations was studied using rotationalviscometry using a Bohlin VOR rheometer (Measure system PP 30, Gap 1.00mm). The results are presented in FIG. 3. MAP counteracts the effect onthe viscosity of the composition caused by the local anesthetic uponsterilization by heat.

Example 4. Non-Crosslinked Hyaluronic Acid with Lidocaine and MAP atLower Concentrations

Formulations as outlined in Table 4 were prepared essentially accordingto the method described in Example 3, with the exception that lowerconcentrations of MAP were used.

TABLE 4 Zero shear Formulation HA Lidocaine MAP viscosity # [mg/ml][mg/ml] [mg/ml] η₀ [Pas] 4a 20 0 0 5.13 4b 20 3 0 6.16 4c 20 3 0.03 5.274d 20 3 0.01 5.87 4e 20 3 0.005 5.91

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The viscosity of the formulations was studied using rotationalviscometry using a Bohling VOR rheometer (Measure system PP 30, Gap 1.00mm).

The results are presented in FIG. 4. MAP counteracts the effect on theviscosity of the composition caused by the local anesthetic uponsterilization by heat.

Example 5. Hyaluronic Acid Gel with Lidocaine and MAP Autoclaved atDifferent F₀-Values

Formulations as outlined in Table 5 were prepared essentially accordingto the method described in Example 1, with the exception that adifferent concentration of MAP was used.

TABLE 5 G′ Formulation HA Lidocaine MAP Average at 1.0 Hz # [mg/ml][mg/ml] [mg/ml] F₀ [Pa] 5a 20 0 0 22 194 5b 20 3 0 22 269 5c 20 3 0.3 22220 5d 20 0 0  6 317 5e 20 3 0  6 363 5f 20 3 0.3  6 332

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave at the differentF₀-values described in Table 5.

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 5. The effect on the elastic modulusG′ of the composition caused by the local anesthetic upon sterilizationby heat is slightly larger for the higher F₀-value. MAP counteracts theeffect on the elastic modulus G′ of the composition caused by the localanesthetic upon sterilization by heat.

Example 6. Hyaluronic Acid Gel with Bupivacaine and MAP

Formulations as outlined in Table 6 were prepared essentially accordingto the method described in Example 1, with the exceptions that lidocainewas replaced by bupivacaine (CAS-number 2180-92-9, Cambrex, Karlskoga,Sweden) and that a hyaluronic acid gel with a modification degree of<1%, with a hyaluronic acid content of 12 mg/ml was used.

G′ Formulation HA Gel Bupivacaine MAP at 1.0 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 6a 12 0 0 62 6b 12 1 0 90 6c 12 1 0.3 61

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 6. Bupivacaine has similar effect onthe elastic modulus G′ of the composition as lidocaine. MAP counteractsthe effect on the elastic modulus G′ of the composition caused by thelocal anesthetic upon sterilization by heat.

Example 7. Hyaluronic Acid Gel with Tetracaine and MAP

Formulations as outlined in Table 7 were prepared essentially accordingto the method described in Example 1 with the exception that lidocainewas replaced by tetracaine (CAS-number 136-47-0, Sigma Aldrich, StLouis, USA) and the concentration of MAP was 0.3 mg/ml.

TABLE 7 G′ Formulation HA Gel Tetracaine MAP at 0.1 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 7a 20 0 0 154 7b 20 3 0 237 7c 20 3 0.3 196

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 7. Tetracaine has similar effect onthe elastic modulus G′ of the composition as lidocaine. MAP counteractsthe effect on the elastic modulus G′ of the composition caused by thelocal anesthetic upon sterilization by heat.

Example 8. Hyaluronic Acid Gel with Lidocaine and SAP

Formulations as outlined in Table 10 were prepared essentially accordingto the method described in Example 1, with the exception that MagnesiumAscorbyl Phosphate (MAP) was replaced by Sodium Ascorbyl Phosphate(SAP).

A stock-solution of SAP (CAS number 66170-10-3, Sigma Aldrich, St Louis,USA) was prepared by dissolving SAP in phosphate buffered saline (8 mM,0.9% NaCl).

TABLE 8 G′ Formulation HA Gel Lidocaine SAP at 0.1 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 8a 20 0 0 285 8b 20 3 0 430 8c 20 3 0.07 374

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜29).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 8. SAP counteracts the effect on theelastic modulus G′ of the composition caused by the local anestheticupon sterilization by heat.

Example 9. Hyaluronic Acid Gel with Lidocaine and MethylsilanolAscorbate

Formulations as outlined in Table 11 were prepared essentially accordingto the method described in Example 1, with the exceptions that MagnesiumAscorbyl Phosphate (MAP) was replaced by Ascorbosilane C (product number078, Exsymol, Monaco) that contains methylsilanol ascorbate (CAS number187991-39-5).

TABLE 9 Methylsilanol G′ Formulation HA Gel Lidocaine ascorbate at 1.0Hz # [mg/ml] [mg/ml] [mg/ml] [Pa] 9a 20 0 0 194 9b 20 3 0 269 9c 20 30.3 134

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 9. Methylsilanol ascorbate effectivelycounteracts the effect on the elastic modulus G′ of the compositioncaused by the local anesthetic upon sterilization by heat.

Example 10. Non-Crosslinked Hyaluronic Acid with Bupivacaine andAscorbyl Glucoside

Formulations as outlined in Table 10 were prepared essentially accordingto the method described in Example 3, with the exception that lidocainewas replaced by bupivacaine and MAP was replaced by ascorbyl glucoside(CAS number 129499-78-1, CarboMer, Inc, San Diego, USA).

TABLE 10 Zero Ascorbyl shear Formulation HA Bupivacaine glucosideviscosity # [mg/ml] [mg/ml] [mg/ml] η₀ [Pas] 10a 20 0 0 1.79 10b 20 1 02.34 10c 20 1 5 2.11

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The viscosity of the formulations was studied using rotationalviscometry using a Bohling VOR rheometer (Measure system C14).

The results are presented in FIG. 10. Ascorbyl glucoside counteracts theeffect on the viscosity of the composition caused by the localanesthetic upon sterilization by heat.

Example 11. Hyaluronic Acid Gel with Lidocaine and DifferentConcentrations of SAP

Formulations as outlined in Table 11 were prepared essentially accordingto the method described in Example 8, with the exception that differentconcentrations of Sodium Ascorbyl Phosphate, SAP were used.

TABLE 11 G′ Formulation HA Lidocaine SAP at 1.0 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 11a 20 0 0 159 11b 20 3 0 290 11c 20 3 0.005 287 11d 20 30.1 256 11e 20 3 0.5 175

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR. Theresults are presented in FIG. 11. SAP counteracts the effect on theelastic modulus G′ of the composition caused by the local anestheticupon sterilization by heat. The higher the concentration of SAP thegreater is the effect.

Example 12. Hyaluronic Acid Gel with Tetracaine and L-Ascorbic AcidAcetonide

Formulations as outlined in Table 12 were prepared essentially accordingto the method described in Example 7 with the exceptions that MAP wasreplaced by L-ascorbic acetonide (CAS-number 15042-01-0, Carbosynth,Berkshire, UK) and a higher concentration of the derivative was used.

TABLE 12 L-Ascorbic G′ Formulation HA Gel Tetracaine acid acetonide at1.0 Hz # [mg/ml] [mg/ml] [mg/ml] [Pa] 12a 20 0 0 266 12b 20 3 0 345 12c20 3 1.0 25

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜5).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 12. L-Ascorbic acetonide effectivelycounteracts the effect on the elastic modulus G′ of the compositioncaused by the local anesthetic upon sterilization by heat.

Example 13. Hyaluronic Acid Gel with a Higher Degree of Modificationwith Lidocaine and SAP

Formulations as outlined in Table 13 were prepared essentially accordingto the method described in Example 1, with the exceptions that ahyaluronic acid gel with a higher degree of modification (approximately7%) was used, that Magnesium Ascorbyl Phosphate (MAP) was replaced bySodium Ascorbyl Phosphate, SAP (CAS number 66170-10-3, Sigma Aldrich, StLouis, USA), and that another concentration of the derivative was used.

TABLE 13 G′ Formulation HA Gel Lidocaine SAP at 1.0 Hz # [mg/ml] [mg/ml][mg/ml] [Pa] 13a 20 0 0 1110 13b 20 3 0 1260 13c 20 3 0.1 1150

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜32).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 13. SAP counteracts the effect on theelastic modulus G′ of the composition caused by the local anestheticupon sterilization by heat.

Example 14. Non-Crosslinked Hyaluronic Acid with Lidocaine andAminopropyl Ascorbyl Phosphate

Formulations as outlined in Table 14 were prepared essentially accordingto the method described in Example 3, with the exceptions that MagnesiumAscorbyl Phosphate (MAP) was replaced by Aminopropyl Ascorbyl Phosphate(Macro Care, South Korea) and that a higher concentration of thederivative was used.

TABLE 14 Aminopropyl Zero Ascorbyl shear Formulation HA Lidocainephosphate viscosity # [mg/ml] [mg/ml] [mg/ml] η₀ [Pas] 14a 20 0 0 2.2914b 20 3 0 3.45 14c 20 3 1.5 1.76

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The viscosity of the formulations was studied using rotationalviscometry using a Bohling VOR rheometer (Measure system PP 30, Gap 1.00mm).

The results are presented in FIG. 14. Aminopropyl Ascorbyl phosphateeffectively counteracts the effect on the viscosity of the compositioncaused by the local anesthetic upon sterilization by heat.

Example 15. Hyaluronic Acid Gel with Lidocaine and Ascorbyl Glucoside

Formulations as outlined in Table 15 were prepared essentially accordingto the method described in Example 1 with the exceptions that MagnesiumAscorbyl Phosphate (MAP) was replaced by Ascorbyl glucoside (CarboMer,Inc, San Diego, USA) and another concentration of the derivative wasused.

TABLE 15 Ascorbyl G′ Formulation HA Gel Lidocaine glucoside at 1.0 Hz #[mg/ml] [mg/ml] [mg/ml] [Pa] 15a 20 3 0 833 15b 20 3 0.08 777

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜23).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 15. Ascorbyl glucoside counteracts theeffect on the elastic modulus G′ of the composition caused by the localanesthetic upon sterilization by heat.

Example 16. Hyaluronic Acid Gel with Lidocaine and Ascorbyl Glucoside

Formulations as outlined in Table 16 were prepared essentially accordingto the method described in Example 15 with the exceptions that ahyaluronic acid gel with a modification degree of <1%, with a hyaluronicacid content of 12 mg/ml was used and that a higher concentration of thederivative was used.

TABLE 16 Ascorbyl G′ Formulation HA Gel Lidocaine glucoside at 1.0 Hz #[mg/ml] [mg/ml] [mg/ml] [Pa] 16a 12 3 0 84 16b 12 3 0.17 80

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜23).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 16. Ascorbyl glucoside counteracts theeffect on the elastic modulus G′ of the composition caused by the localanesthetic upon sterilization by heat.

Example 17. Hyaluronic Acid Gel with Lidocaine and Ascorbyl Glucoside

Formulations as outlined in Table 17 were prepared essentially accordingto the method described in Example 15 with the exceptions that Ascorbylglucoside from another manufacturer (Hayashibara BiochemicalLaboratories, Inc, Okayama, Japan) was used and that higherconcentrations of the derivative were used. In this example a hyaluronicacid gel with a hyaluronic acid content of 16 mg/ml was used.

TABLE 17 Ascorbyl G′ Formulation HA Gel Lidocaine glucoside at 1.0 Hz #[mg/ml] [mg/ml] [mg/ml] [Pa] 17a 16 3 0 330 17b 16 3 0.8 314 17c 16 38.0 301

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜23).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 17. Ascorbyl glucoside counteracts theeffect on the elastic modulus G′ of the composition caused by the localanesthetic upon sterilization by heat.

Example 18. Hyaluronic Acid Gels with Different Degrees of Modificationwith Lidocaine and MAP

Formulations as outlined in Table 18 were prepared essentially accordingto the method described in Example 1 with the exception that anotherconcentration of MAP was used. In this example hyaluronic acid gels withdifferent degrees of modification were used.

TABLE 18 G′ HA at For- Gel/ Degree of 1.0 Reduction mulation Solutionmodification Lidocaine MAP Hz in G′ # [mg/ml] [mole %] [mg/ml] [mg/ml][Pa] [%] 18a 20 <1 3 0  66 — 18b 20 <1 3 0.3  38 43 18c 20 1 3 0 269 —18d 20 1 3 0.3 220 18 18e 20 7 3 0 417 — 18f 20 7 3 0.3 388 7

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 18. MAP counteracts the effect on theelastic modulus G′ of the composition caused by the local anestheticupon sterilization by heat. The effect is more pronounced in theformulations with a lower degree of modification.

Example 19. Stability Study for 14 Days in 60° C. Hyaluronic Acid Gelwith a Higher Degree of Modification with Lidocaine and SAP

Formulations as outlined in Table 19 were prepared essentially accordingto the method described in Example 2, with the exceptions that MagnesiumAscorbyl Phosphate (MAP) was replaced by Sodium Ascorbyl Phosphate, SAPand that a lower concentration of the derivative was used.

TABLE 19 Formulation HA Gel Lidocaine SAP # [mg/ml] [mg/ml] [mg/ml] 19a20 0 0 19b 20 3 0 19c 20 3 0.1

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜32).

A stability study in 60° C. for 14 days was performed with sampling at0, 3, 7, 11 and 14 days.

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 19. The stability of the compositionis not increased by SAP. The degradation rate of the composition withSAP corresponds to the composition without SAP.

Example 20. Stability Study for 14 Days in 60° C. Hyaluronic Acid Gelwith Lidocaine and Ascorbyl Glucoside

Formulations as outlined in Table 20 were prepared essentially accordingto the method described in Example 1, with the exceptions that MagnesiumAscorbyl Phosphate (MAP) was replaced by Ascorbyl glucoside (CarboMer,Inc, San Diego, USA) and that another concentration of the derivativewas used.

TABLE 20 Ascorbyl Formulation HA Gel Lidocaine glucoside # [mg/ml][mg/ml] [mg/ml] 20a 20 3 0 20b 20 3 0.17

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜22).

A stability study in 60° C. for 14 days was performed with sampling at0, 7 and 14 days.

The gel content was determined by adding an excess of saline to a knownamount of the preparation and dispersing the gel thoroughly to form adilute suspension. The diluted suspension of the gel was filteredthrough a 0.22 mm filter and the concentration of HA in the filtrate,“the extractable part”, was determined using the carbazol method. Thegel content was calculated as the fraction of HA in the filler thatcannot pass through the 0.22 mm filter when filtering the dilutedsuspension of the product.

The results are presented in FIG. 20. There is no change in stabilityfor the composition with Ascorbyl glucoside compared to the formulationwithout Ascorbyl glucoside.

Example 21. Stability Study for 14 Days in 60° C. Hyaluronic Acid Gelwith Lidocaine, MAP or Ascorbyl Glucoside

Formulations as outlined in Table 21 were prepared essentially accordingto the method described in Example 1, with the exceptions that MAP orAscorbyl glucoside (CarboMer, Inc, San Diego, USA) were used and that ahyaluronic acid gel with a modification degree of <1%, with a hyaluronicacid content of 12 mg/ml was used.

TABLE 21 Ascorbyl Formulation HA Gel Lidocaine MAP glucoside # [mg/ml][mg/ml] [mg/ml] [mg/ml] 21a 12 3 0 0 21b 12 3 0.07 0 21c 12 3 0 0.07

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜26).

A stability study in 60° C. for 14 days was performed with sampling at0, 3, 7, 11 and 14 days.

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 21. The stability of the compositionis unaffected by Ascorbyl glucoside. In the composition with MAP aslight decrease in the stability is seen. However, the inventors havefound that the stability of the compositions is still acceptable andthat advantages associated with adding the ascorbic acid derivativeoutweigh the slight decrease in stability caused by the addition.

Example 22. Stability Study for 16 Hours in 90° C. Hyaluronic Acid Gelwith Lidocaine and Ascorbyl Glucoside

Formulations as outlined in Table 22 were prepared essentially accordingto the method described in Example 1, with the exceptions that MagnesiumAscorbyl Phosphate (MAP) was replaced by Ascorbyl glucoside (HayashibaraBiochemical Laboratories, Inc, Okayama, Japan) and that otherconcentrations of the derivative were used. In this example a hyaluronicacid gel with a hyaluronic acid content of 16 mg/ml was used.

TABLE 22 Ascorbyl Formulation HA Gel Lidocaine glucoside # [mg/ml][mg/ml] [mg/ml] 22a 16 3 0 22b 16 3 0.17 22c 16 3 8.0

The pH values of the formulations were adjusted to 7.5±0.2 and theformulations were filled in 1 ml glass syringes (BD Hypak SCF) andautoclaved in a Getinge GEV 6610 ERC-1 autoclave (F₀˜26).

A stability study in 90° C. for 16 hours was performed with sampling at0, 8 and 16 hours.

The rheological properties of the formulations were analysed using aBohlin VOR Reometer (Measure system PP 30, Gap 1.00 mm). Initially astrain sweep was made to find the linear viscoelastic region (LVR) andthen the viscoelastic properties were measured within the LVR.

The results are presented in FIG. 22. Ascorbyl glucoside in the lowerconcentration does not affect the stability of the composition. Thehigher concentration of Ascorbyl glucoside decreases the stability ofthe composition. From these results it was concluded a concentration ofAscorbyl glucoside of below 5 mg/ml is preferred, since higherconcentrations may result in unnecessary decrease of stability of thehyaluronic acid composition.

The invention claimed is:
 1. A sterilized injectable hyaluronic acidcomposition, comprising: a hyaluronic acid gel, a therapeuticallyrelevant concentration of lidocaine, and an ascorbic acid derivativeselected from the group consisting of ascorbyl phosphates, ascorbylsulfates, and ascorbyl glycosides, in an amount which prevents orreduces the effect on the viscosity and/or elastic modulus G′ of thecomposition caused by the lidocaine upon sterilization by heat, whereinthe lidocaine is present in a concentration in the range of 1 to 5mg/ml, wherein the concentration of the ascorbic acid derivative in thecomposition is in the range of 0.01 to 5 mg/ml, and the composition hasbeen subjected to sterilization by autoclaving at a F0-value≥4, whereinthe hyaluronic acid gel is crosslinked by modification with a chemicalcrosslinking agent, wherein a degree of modification of the hyaluronicacid gel is less than 2 mol %, and wherein the hyaluronic acidcomposition does not exhibit increased stability compared to the samecomposition without an ascorbic acid derivative.
 2. The injectablehyaluronic acid composition according to claim 1, wherein the ascorbicacid derivative is selected from the group consisting of ascorbylphosphates and ascorbyl glycosides, or a combination thereof.
 3. Theinjectable hyaluronic acid composition according to claim 2, wherein theascorbic acid derivative is an ascorbyl glycoside.
 4. The injectablehyaluronic acid composition according to claim 2, wherein the ascorbicacid derivative is ascorbyl glucoside.
 5. The injectable hyaluronic acidcomposition according to claim 1, wherein the concentration of theascorbic acid derivative is in the range of 0.01 to 0.5 mg/ml.
 6. Theinjectable hyaluronic acid composition according to claim 2, wherein theascorbic acid derivative is selected from the group consisting of sodiumascorbyl phosphate (SAP) and magnesium ascorbyl phosphate (MAP).
 7. Theinjectable hyaluronic acid composition according to claim 6, wherein theconcentration of the sodium ascorbyl phosphate (SAP) or magnesiumascorbyl phosphate (MAP) is in the range of 0.01 to 1 mg/ml.
 8. Theinjectable hyaluronic acid composition according to claim 7, wherein theconcentration of the sodium ascorbyl phosphate (SAP) or magnesiumascorbyl phosphate (MAP) is in the range of 0.01 to 0.5 mg/ml.
 9. Theinjectable hyaluronic acid composition according to claim 4, wherein theconcentration of the ascorbyl glucoside is in the range of 0.01 to 1mg/ml.
 10. The injectable hyaluronic acid composition according to claim9, wherein the concentration of the ascorbyl glucoside is in the rangeof 0.01 to 0.8 mg/ml.
 11. The injectable hyaluronic acid compositionaccording to claim 10, wherein the concentration of the ascorbylglucoside is in the range of 0.05 to 0.4 mg/ml.
 12. A medicamentcomprising the injectable hyaluronic acid composition according to claim1.